The primary goal of this project is to obtain information about community function by focusing on the critical initial oxygenases that are the rate-limiting step in productive degradation and eventual mineralization of aromatic compounds in oxic and suboxic environments. This project will explore the diversity of oxygenase gene sequences and monitor shifts in their population without regard to host organism. Results of the proposed experiments will identify the breadth of genetic diversity of oxygenases in the environment and associate that diversity with known and unknown organisms. In particular the data will identify gene targets for better molecular assessment of hydrocarbon oxidation in the environment. From this two-year exploratory research program we anticipate the development of a large database of oxygenase gene sequences. It is anticipated that the majority of these will be novel sequences, derived from organisms that are not yet in laboratory cultivation. Our ability to successfully obtain such sequences will result from the use of novel oxygenase PCR primers. Full length oxygenase gene sequences will be obtained through inverse PCR and sequencing of environmental DNA cosmid clones. The goal of developing and using these molecular approaches is to assess their robustness for tracking changes in oxygenase gene populations in laboratory-scale environmental perturbation experiments or in remediation treatment applications. This will be accomplished by focusing intensively on source materials from a single Superfund site in which differences in physical, chemical and geological site parameters can be assessed for their impact on the "molecular fingerprints" of the microbial oxygenase patterns that result from the experimental treatments. Data from this type of exploratory study should allow us to determine which molecular signatures (in this case, dioxygenase patterns) are predictive of overall biodegradative perforrnance of the system. From this, we plan to construct a new generation of "oxygenase microarray" that can provide functional information about those members of the microbial community that become important in biodegradative and bioremediative processes.